Sustained-release preparation

ABSTRACT

A sustained-release preparation which comprises a physiologically active peptide of general formula:                    
     wherein X represents an acyl group; 
     R 1 , R 2  and R 4  each represents an aromatic cyclic group; 
     R 3  represents a D-amino acid residue or a group of the formula:                    
     wherein R 3 ′ is a heterocyclic group; 
     R 5  represents a group of the formula —(CH 2 ) n —R 5 ′ wherein n is 2 or 3, and R 5 ′ is an amino group which may optionally be substituted, an aromatic cyclic group or an O-glycosyl group; 
     R 6  represents a group of the formula —(CH 2 ) n —R 6 ′ wherein n is 2 or 3, and R 6 ′ is an amino group which may optionally be substituted; 
     R 7  represents a D-amino acid residue or an azaglycyl residue; and 
     Q represents hydrogen or a lower alkyl group, or a salt thereof and a biodegradable polymer having a terminal carboxyl group. 
     The sustained-release preparation shows a constant release of the peptide over a long time and is substantially free from an initial burst.

This application is a divisional of application Ser. No. 08/892,315,filed Jul. 14, 1997 now U.S. Pat. No. 5,972,891, which is a divisionalof application Ser. No. 08/471,382, filed Jun. 6, 1995, now U.S. Pat.No. 5,668,111 which is a divisional of application Ser. No. 08/162,698,filed Dec. 7, 1993 now U.S. Pat. No. 5,480,868.

The present invention relates to a sustained-release preparationcontaining a physiologically active peptide and to a method of producingthe same.

BACKGROUND OF THE INVENTION

The prior art includes, as disclosed in EP-A-481,732, asustained-release preparation comprising a drug, a polylactic acid and aglycolic acid-hydroxycarboxylic acid [HOCH(C₂₋₈ alkyl)COOH] copolymer.The disclosed process comprises preparing a W/O emulsion consisting ofan internal water phase comprising an aqueous solution of aphysiologically active peptide and an external oil phase comprising asolution of a biodegradable polymer in an organic solvent, adding saidW/o emulsion to water or an aqueous medium and processing the resultingW/O/W emulsion into sustained-release microcapsules (drying-in-watermethod).

EP-A-52510 describes a microcapsule comprising a hormonally activepolypeptide, a biodegradable polymer and a polymer hydrolysis controlagent. The disclosed, process for its production is a coacervationprocess which comprises adding a coacervation agent to a W/O emulsionconsisting of an aqueous solution of the polypeptide as the internalwater phase and a halogenated organic solvent as the oil phase toprovide microcapsules.

GB-A-2209937 describes a pharmaceutical composition comprising apolylactide, a polyglycolide, a lactic-acid-glycolic acid copolymer or amixture of these polymers and a water-insoluble peptide. Also disclosedis a production process which comprises dispersing a salt of thewater-insoluble peptide in a solution of said polylactide,polyglycolide, a lactic acid-glycolic acid copolymer or a mixture ofthese polymers, removing the solvent by evaporation and molding theresulting mixture into solid particles.

EP-A-58481 describes a process for producing a pharmaceuticalcomposition comprising a polylactide and an acid-stable polypeptidewhich, for instance, comprises dissolving tetragastrin hydrochloride anda polylactide in aqueous dioxane, casting the solution into a film andevaporating the solvent.

EP-A-0467389 teaches a technology for providing a drug delivery systemfor proteins and polypeptides by the polymer precipitation technique orthe microsphere technique. However, this literature contains no specificdisclosure about a system containing an LH-RH derivative.

The luteinizing hormone-releasing hormone, known as LH-RH (or GnRH), issecreted from the hypothalamus and binds to receptors on the pituitarygland. The LH (luteinizing hormone) and FSH (follicle stimulatinghormone), which are released thereon, act on the gonad to synthesizesteroid hormones. As derivatives of LH-RH, the existence of bothagonistic and antagonistic peptides is known. When a highly agonisticpeptide is repeatedly administered, the available receptors are reducedin number so that the formation of gonad-derived steroidal hormones issuppressed. Therefore, LH-RH derivatives are expected to be of value astherapeutic agents for hormone-dependent diseases such as prostatecancer, benign prostatomegaly, endometriosis, hysteromyoma,metrofibroma, precocious puberty, mammary cancer, etc. or ascontraceptives. Particularly, the problem of histamine-releasingactivity was pointed out for LH-RH antagonists of the so-called firstand second generations (The Pharmaceuticals Monthly 32, 1599-1605, 1990)but a number of compounds have since been synthesized and recentlyLH-RH-antagonizing peptides having no appreciable histamine-releasingactivity have been developed (cf. U.S. Pat. No. 5,110,904, forinstance). In order for any such LH-RH antagonizing peptide to manifestits pharmacological effect, there is a need for a controlled releasesystem so that the competitive inhibition of endogenous LH-RH may bepersistent. Moreover, because of histamine-releasing activity which maybe low but is not non-existent in such peptides, a demand exists for asustained-release preparation with an inhibited initial burstimmediately following administration.

Particularly, in the case of a sustained-release (e.g. 1-3 months)preparation, it is important to insure a more positive and constantrelease of the peptide in order that the desired efficacy may beattained with greater certainty and safety.

At the same time, there is a long-felt need for a method of producing asustained-release preparation having a high peptide trap rate for aphysiologically active peptide, particularly LH-RH-antagonizingpeptides.

SUMMARY OF THE INVENTION

According to the present invention, there is provided:

1) A sustained-release preparation which comprises a physiologicallyactive peptide of the general formula:

wherein

X represents an acyl group;

R₁, R₂ and R₄ each represents an aromatic cyclic group;

R₃ represents a D-amino acid residue or a group of the formula:

wherein

R₃′ is a heterocyclic group;

R₅ represents a group of the formula —(CH₂)_(n)—R₅′ wherein n is 2 or 3,and R₅′ is an amino group which may optionally be substituted, anaromatic cyclic group or an O-glycosyl group;

R₆ represents a group of the formula —(CH₂)_(n)—R₆′ wherein n is 2 or 3,and R₆′ is an amino group which may optionally be substituted;

R₇ represents a D-amino acid residue or an azaglycyl residue; and

Q represents hydrogen or a lower alkyl group or a salt thereof and abiodegradable polymer having a terminal carboxyl group,

2) The sustained-release preparation according to the above paragraph 1,wherein X is a C₂₇ alkanoyl group which may optionally be substituted bya 5- or 6-membered heterocyclic carboxamido group,

3) The sustained-release preparation according to the above paragraph 2,wherein X is a C₂₋₄, alkanoyl group which may optionally be substitutedby a tetrahydrofurylcarboxamide group,

4) The sustained-release preparation according to the above paragraph 1,wherein X is acetyl,

5) The sustained-release preparation according to the above paragraph 1,wherein the biodegradable polymer is a mixture of (A) a copolymer ofglycolic acid and a hydroxycarboxylic acid of the general formula:

 wherein R represents an alkyl group of 2 to 8 carbon atoms and (B) apolylactic acid,

6) The sustained-release preparation according to the above paragraph 1,wherein X is acetyl, and the biodegradable polymer is a mixture of (A) acopolymer of glycolic acid and a hydroxycarboxylic acid of the generalformula (III and (B) a polylactic acid,

7) The sustained-release preparation according to the above paragraph 5,wherein the copolymer has a weight average molecular weight of about2,000 to 50,000, as determined by GPC,

8) The sustained-release preparation according to the above paragraph 5,wherein the copolymer has a dispersion value of about 1.2 to 4.0,

9) The sustained-release preparation according to the above paragraph 5,wherein the polylactic acid has a weight average molecular weight ofabout 1,500 to 30,000 as determined by GPC,

10) The sustained-release preparation according to the above paragraph5, wherein the polylactic acid has a dispersion value of about 1.2 to4.0,

11) The sustained-release preparation according to the above paragraph1, wherein the biodegradable polymer Ls a copolymer of lactic acid andglycolic acid,

12) The sustained-release preparation according to the above paragraph11, wherein the copolymer has a weight average molecular weight of about5,000 to 25,000, as determined by GPC,

13) The sustained-release preparation according to the above paragraph11, wherein the copolymer has a dispersion value of about 1.2 to 4.0,

14) The sustained-release preparation according to the above paragraph1, wherein the proportion of the physiologically active peptide rangesfrom about 0.01 to 50% (w/w) based on the biodegradable polymer,

15) The sustained-release preparation according to the above paragraph1, wherein the physiologically active peptide is a LH-RH antagonist,

16) The sustained-release preparation according to the above paragraph1, wherein the physiologically active peptide is:

 or its acetate,

17) The sustained-release preparation according to the above paragraph1, wherein the physiologically active peptide isNAcD2Nal-D4ClPhe-D3Pal-ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂ orits acetate,

18) The sustained-release preparation according to the above paragraph1, wherein the physiologically active peptide isNAcD2Nal-D4ClPhe-D3Pal-ser-Tyr-DhArg(Et₂)-Leu-hArg(Et₂)-Pro-DAlaNH₂ orits acetate,

19) A method of producing a sustained-release preparation whichcomprises dissolving a physiologically active peptide of the generalformula [I] or a salt thereof and a biodegradable polymer having aterminal carboxyl group in a solvent which is substantially immisciblewith water and then removing said solvent,

20) The method according to the above paragraph 19, wherein thebiodegradable polymer is a mixture of (A) a copolymer of glycolic acidand a hydroxycarboxylic acid of the general formula [II] and (B) apolylactic acid,

21) The method according to the above paragraph 19, wherein X is acetyl,and the biodegradable polymer is a mixture of (A) a copolymer ofglycolic acid and a hydroxycarboxylic acid of the general formula [II]and (B) a polylactic acid,

22) The method according to the above paragraph 19, wherein thebiodegradable polymer is a copolymer of lactic acid and glycolic acid,

23) A method according to the above paragraph 19, which comprisesdissolving the biodegradable polymer and the physiologically activepeptide in a solvent which is substantially immiscible with water andadding the resulting solution to an aqueous medium to provide an O/Wemulsion,

24) A method of producing a sustained-release preparation whichcomprises dissolving a biodegradable polymer comprising a mixture of (A)a copolymer of glycolic acid and a hydroxycarboxylic acid of the generalformula:

 wherein R represents an alkyl group of 2 to 8 carbon atoms and (B) apolylactic acid and a substantially water-insoluble physiologicallyactive peptide or a salt thereof in a solvent which is substantiallyimmiscible with water and then removing said solvent, and

25) A method according to the above paragraph 24, which furthercomprises after dissolving the biodegradable polymer and thesubstantially water-insoluble peptide or salt thereof in the solventadding the resulting solution to an aqueous medium to provide an O/Wemulsion.

DETAILED DESCRIPTION OF THE INVENTION

The abbreviations used in this specification have the followingmeanings.

NAcD2Nal: N-Acetyl-D-3-(2-naphtyl)alanyl

D4ClPhe: D-3-(4-Chlorophenyl)alanyl

D3Pal: D-3-(3-Pyridyl)alanyl

NMeTyr: N-Methylthyrosyl

DLys(Nic): D-(Ipsilon-N-nicotinoyl)lysyl

tys(Nisp): (Ipsilon-N-isopropyl)lysyl

DLys(AzaglyNic): D-[1-Aza-(N-nicotinoyl)glycyl]lysyl

DLys(AzaglyFur): D-[1-Aza-(N-2-furoyl)glycyl]lysyl

Where any other amino acids are expressed by abbreviations, theabbreviations recommended by IUPAC-IUB Commission on BiochemicalNomenclature (European Journal of Biochemistry 138, 9-37, 1984) or theabbreviations in common usage in the art are used. Where optical isomersexist for any compound, the L-isomer is meant unless otherwiseindicated.

In the present invention, the peptide [I] shows LH-RH antagonisticactivity and is effective for the treatment of hormone-dependentdiseases such as prostatic cancer, prostatomegaly, endometriosis,hysteromyoma, metrofibroma, precocious puberty, mammary cancer, etc. orfor contraception.

Referring to general formula [I], the acyl group X is preferably an acylgroup derived from carboxylic acid. Examples of the acyl group include aC₂₋₇ alkanoyl, C₇₋₁₅ cycloalkenoyl (e.g., cyclohexenoyl), C₁₋₆alkylcarbamoyl (e.g., ethyl carbamoyl), 5- or 6-membered heterocycliccarbonyl (e.g. piperidinocarbonyl) and carbamoyl group which mayoptionally be substituted. The acyl group is preferably a C₂₋₇ alkanoylgroup (e.g., acetyl, propionyl, butyryl, isobutyryl, pentanoyl, hexanoylor heptanoyl) which may optionally be substituted, more preferably C₂₋₄alkanoyl group (e.g., acetyl, propionyl, butyryl, isobutyryl) which mayoptionally be substituted. The substituents are for example C₁₋₆alkylamino group (e.g., methylamino, ethylamino, diethylamino,propylamino), C₁₋₃ alkanoyl amino group (e.g., formylamino, acetylamino,propionylamino), C₇₋₁₅ cycloalkenoyl amino group (e.g.,cyclohexenoylamino), C₇₋₁₅ arylcarbonyl-amino group (e.g.,benzoylamino), 5- or 6-membered heterocyclic carboxamido group (e.g.,tetrahydrofurylcarboxamido, pyridylcarboxamido, furylcarboxamido),hydroxyl group, carbamoyl group, formyl group, carboxyl group, 5- or6-membered heterocyclic group (e.g., pyridyl, morpholino). Thesubstituents are preferably 5- or 6-membered heterocyclic carboxamidogroup (e.g., tetrahydrofurylcarboxamido, pyridylcarboxamido,furylcarboxamido).

X is preferably a C₂₋₇ alkanoyl group which may optionally besubstituted by a 5- or 6-membered heterocyclic carboxamido group.

X is more preferably a C₂₋₄ alkanoyl group which may optionally besubstituted by a tetrahydrofuryl carboxamido group.

Specific examples of X are acetyl,

and so on.

The aromatic cyclic group R₁, R₂ or R₄ may for example be an aromaticcyclic group of 6 to 12 carbon atoms. Examples of the aromatic cyclicgroup are phenyl, naphthyl, anthryl and so on. Preferred are aromaticcyclic groups of 6 to 10 carbon atoms, such as phenyl and naphthyl.These aromatic cyclic groups may each have 1 to 5, preferably 1 to 3,suitable substituents in appropriate positions on the ring. Suchsubstituents include hydroxyl, halogen, aminotriazolyl-substitutedamino, alkoxy and so on. Preferred are hydroxy, halogen andaminotriazolyl-substituted amino.

The halogens mentioned above include fluorine, chlorine, bromine andiodine.

The aminotriazolyl moiety of said aminotriazolyl-substituted aminoincludes, among others, 3-amino-1H-1,2,4-triazol-5-yl,5-amino-1H-1,3,4-triazol-2-yl, 5-amino-1H-1,2,4-triazol-3-yl,3-amino-2H-1,2,4-triazol-5-yl, 4-amino-1H-1,2,3-triazol-5-yl,4-amino-2H-1,2,3-triazol-5-yl and so on.

The alkoxy group is preferably an alkoxy group of 1 to 6 carbon atoms(e.g. methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, etc.).

More preferably, R₁ is naphthyl or halophenyl. More preferably, R₂ ishalophenyl. More preferably, R₄ is hydroxyphenyl oraminotriazolylamino-substituted phenyl.

The D-amino acid residue R₃ is preferably an α-D-amino acid residue of 3to 12 carbon atoms. Examples of the amino acid are leucine, isoleucine,norleucine, valine, norvaline, 2-aminobutyric acid, phenylalanine,serine, threonine, methionine, alanine, tryptophan and aminoisobutyricacid. These amino acids may have suitable protective groups (theprotective groups used conventionally in the art, such as t-butyl,t-butoxy, t-butoxycarbonyl, etc.).

The heterocyclic group R₃′ includes 5- or 6-membered heterocyclic groupseach containing 1 to 2 nitrogen or sulfur atoms as hetero-atoms, whichmay optionally be fused to a benzene ring. Specifically, thienyl,pyrrolyl, thiazolyl, isothiazolyl, imidizolyl, pyrazolyl, pyridyl,3-pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 3-benzo[b]thienyl,3-benzo[b]-3-thienyl, indolyl, 2-indolyl, isoindolyl, 1H-indazolyl,benzoimidazolyl, benzothiazolyl, quinolyl, isoquinolyl, etc. may bementioned. The particularly preferred species of R₃′ is pyridyl or3-benzo[b]thienyl.

The aromatic cyclic group R₅ may be the same as the aromatic cyclicgroup R₁, R₂ or R₄. The aromatic cyclic group may have 1 to 5.preferably 1 to 3, suitable substituents in appropriate positions on thering. The substituents may also be the same as the substituentsmentioned for R₁, R₂ or R₄. The particularly preferred substituent isaminotriazolyl-substituted amino.

The glycosyl group for O-glycosyl R₅ is preferably a hexose or aderivative thereof. The hexose includes D-glucose, D-fructose,D-mannose, D-galactose, L-galactose and so on. As said derivative, deoxysugars (L- and D-fucose, D-quinovose, L-rhamnose, etc.) and amino sugars(D-glucosamine, D-galactosamine, etc.) can be mentioned. More preferredare deoxy sugars (L- and D-fucose, D-quinovose, L-rhamnose, etc.). Stillmore preferred is L-rhamnose.

The substituent on the amino group which may optionally be substituted,R₅′, includes, among others, acyl, carbamoyl, carbazoyl which may besubstituted by acyl or amidino which may be mono- or di-substituted byalkyl.

The above-mentioned acyl and the acyl for the above-mentioned carbazoylwhich may be substituted by acyl include nicotinoyl, furoyl, thenoyl andso on.

The alkyl moiety of the mono- or di-alkylamidino mentioned aboveincludes straight-chain or branched alkyl groups of 1 to 4 carbon atoms,thus including methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl and tert-butyl and so on. The preferred alkyl moiety is methylor ethyl.

The substituent for the amino group which may optionally be substituted,R₆′, includes alkyl and amidino which may be mono- or di-substituted byalkyl.

The above-mentioned alkyl and the alkyl of the mono- or dialkylamidinomentioned above include those alkyl groups mentioned for R₅′.

The D-amino acid residue R₇ is preferably a D-amino acid residue of 3 to9 carbon atoms, such as D-alanyl, D-leucyl, D-valyl, D-isoleucyl,D-phenylalanyl and so on. More preferred are D-amino acid residues of 3to 6 carbon atoms, such as D-alanyl, D-valyl and so on. The-morepreferred species of R₇ is D-alanyl.

The lower alkyl group Q may be the alkyl group defined for R₅′. The mostpreferred species of Q is methyl.

Specific examples of R₁ are:

Specific examples of R₂ are:

Specific examples of R₃ are:

Specific examples of R₄ are:

Specific examples of R₅ are:

Specific examples of R₆ are:

Specific examples of R₇ are:

When the peptide [I] has one or more asymmetric carbon atom(s), thereare two or more stereoisomers. Any of such steroisomers as well as amixture thereof is within the scope of the present invention.

The peptide of general formula [I] is produced by the per se knownprocesses. Typical specific processes are described in U.S. Pat. No.5,110,904.

The peptide [I] can be used in the form of a salt, preferably apharmacologically acceptable salt. Where the peptide has basic groupssuch as amino, the salt includes-salts with inorganic acids (e.g.hydrochloric acid, sulfuric acid, nitric acid, etc.) or organic acids(e.g. carbonic acid, hydrogen carbonic acid, succinic acid, acetic acid,propionic acid, trifluoro-acetic acid, etc.). Where the peptide hasacidic groups such as carboxyl, salts with inorganic bases (e.g. alkalimetals such as sodium, potassium, etc. and alkaline earth metals such ascalcium, magnesium, etc.) or organic bases (e.g. organic amines such astriethylamine and basic amino acids such as arginine). The peptide [I]may be in the form of a metal complex compound (e.g. copper complex,zinc complex, etc.). The preferred salts of peptide [I] are salts withorganic acids (e.g. carbonic acid, hydrogen carbonic acid, succinicacid, acetic acid, propionic acid, trifluoroacetic acid, etc.). The mostpreferred is the acetate.

Particularly preferred species of peptide [I] or salt are as follows.

(1)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂ orits acetate

(2)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH₂or its acetate

(3)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂or its acetate

(4)

 or its acetate

(5) NAcD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DhArg(Et₂)-Leu-hArg(Et₂)-Pro-DAlaNH₂or its acetate

In the sustained-release preparation, the proportion of the peptide [I]may vary with the type of peptide, the expected pharmacological effectand duration of effect, among other factors, and may range from about0.01 to about 50% (w/w) based on the biodegradable polymer. Thepreferred range is about 0.1 to about 40% (w/w) and a more preferredrange is about 1 to about 30% (w/w).

The biodegradable polymer having a terminal carboxyl group is nowdescribed.

A biodegradable polymer, about 1 to 3 g, was dissolved in a mixture ofacetone (25 ml) and methanol (5 ml) and using phenolphthalein as theindicator, the carboxyl groups in the solution were quickly titratedwith 0.05N alcoholic potassium hydroxide solution under stirring at roomtemperature (20° C.). The number average molecular weight by end-groupdetermination was then calculated by means of the following equation.

Number average molecular weight by end-group determination=20000×A/B

where A is the mass of biodegradable polymer (g) B is the amount of0.05N alcoholic potassium hydroxide solution (ml) added to react thetitration end-point.

The result of the above calculation is referred to as the number averagemolecular weight by end-group determination.

By way of illustration, taking a polymer having a terminal carboxylgroup as synthesized from one or more α-hydroxy acids by thenon-catalytic dehydrative poly-condensation process as an example, thenumber average molecular weight by end-group determination isapproximately equal to the number average molecular weight found by GPC.In contrast, in the case of a polymer substantially not containing freeterminal carboxyl groups as synthesized from a cyclic dimer by thering-opening polymerization process and using catalysts, the numberaverage molecular weight by end-group determination is by far greaterthan the number average molecular weight by GPC determination. By thisdifference, a polymer having a terminal carboxyl group can be clearlydiscriminated from a polymer having no terminal carboxyl group. Thus,the term ‘biodegradable polymer having a terminal carboxyl group’ isused herein to mean a biodegradable polymer showing a substantialagreement between the number average molecular weight by GPCdetermination and the number average molecular weight by end-groupdetermination.

Whereas the number average molecular weight by end-group determinationis an absolute value, the number average molecular weight by GPCdetermination is a relative value which varies according to analyticaland procedural conditions (such as types of mobile phase and column,reference substance, selected slice width, selected baseline, etc.).Therefore, the two values cannot be numerically correlated bygeneralization. However, the term ‘substantial agreement’ between thenumber average molecular weight by GPC determination and the numberaverage molecular weight by end-group determination means that thenumber average molecular weight found by end-group determination isabout 0.4 to 2 times, more preferably about 0.5 to 2 times, mostpreferably about 0.8 to 1.5 times, the number average molecular weightby GPC determination. The term ‘by far greater’ as used above means thatthe number average molecular weight by end-group determination is abouttwice or greater than the number average molecular weight by GPCdetermination.

The preferred polymer for the purpose of the present invention is apolymer showing a substantial agreement between the number averagemolecular weight by GPC determination and the number average molecularweight by end-group determination.

As specific examples of the biodegradable polymer having a terminalcarboxyl group can be mentioned polymers and copolymers, as well asmixtures thereof, which are synthesized from one or more species ofα-hydroxy acids (e.g. glycolic acid, lactic acid, hydroxybutyric acid,etc.), hydroxydicarboxylic acids (e.g. malic acid etc.),hydroxytricarboxylic acids (e.g. citric acid etc.), etc. by thenon-catalytic dehydrative polycondensation reaction, poly-α-cyanoacrylicesters, polyamino acids (e.g. poly-γ-benzyl-L-glutamic acid etc.),maleic anhydride copolymers (e.g. styrene-maleic acid copolymer etc.)and so on.

The mode of polymerization may be random, block or graft. Where any ofthe above-mentioned α-hydroxy acids, hydroxydicarboxylic acids andhydroxytricarboxylic acids has an optical activity center within themolecule, any of the D-, L- and DL-forms can be employed.

The biodegradable polymer having a terminal carboxyl group is preferablya biodegradable polymer comprising a mixture of (A) a copolymer ofglycolic acid and a hydroxycarboxylic acid of the general formula:

wherein R represents an alkyl group of 2 to 8 carbon atoms and (B) apolylactic acid, or a lactic acid-glycolic acid copolymer.

Referring to the general formula [II], the straight-chain or branchedalkyl group of 2 to 8 carbon atoms, as represented by R, includes, interalia, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl, hexyl,isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl and2-ethylbutyl. Preferred, among them, are straight-chain or branchedalkyls of 2 to 5 carbon atoms. Specifically, ethyl, propyl, isopropyl,butyl and isobutyl are preferred. R is most preferably ethyl.

The hydroxycarboxylic acid of the general formula [II] includes, interalia, 2-hydroxybutyric acid, 2-hydroxyvaleric acid,2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid,2-hydroxyisocaproic acid and 2-hydroxycapric acid. Preferred are2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyricacid and 2-hydroxycaproic acid. The hydroxycarboxylic acid of thegeneral formula [II] is most preferably 2-hydroxybutyric acid. Whilethese hydroxycarboxylic acids may be any of the D-, L- andD,L-compounds, the D-/L-ratio (mol %) is preferably in the range ofabout 75/25 through about 25/75. The more preferred embodiment is ahydroxycarboxylic acid with a D-/L-ratio (mol %) within the range ofabout 60/40 through about 40/60. The most preferred is ahydroxycarboxylic acid with a D-/L-ratio (mol %) within the range ofabout 55/45 through about 45/55.

Referring to the copolymer of glycolic acid and said hydroxycarboxylicacid of the general formula [II] (hereinafter referred to as glycolicacid copolymer), the mode of copolymerization may be random, block orgraft. Preferred are random copolymers.

The hydroxycarboxylic acids of the general formula [II] can be usedalone or in combination.

The preferred proportions of glycolic acid and hydroxycarboxylic acid[II] in said glycolic acid copolymer (A) are about 10 to about 75 mole %of glycolic acid and the balance of hydroxycarboxylic acid. Moredesirably, the copolymer consists of about 20 to about 75 mole % ofglycolic acid and the balance of hydroxycarboxylic acid. Most desirably,the copolymer consists of about 40 to about 70 mole % of glycolic acidand the balance of hydroxycarboxylic acid. The weight average molecularweight of said glycolic acid copolymer may range from about 2,000 toabout 50,000. The preferred range is about 3,000 to about 40,000. Themore preferred range is about 8,000 to about 30000. The dispersion value(weight average molecular weight/number average molecular weight) ispreferably in the range of about 1.2 to about 4.0. Particularlypreferred are copolymers with dispersion values in the range of about1.5 to about 3.5.

The glycolic acid copolymer (A) can be synthesized by the knowntechnology, for example by the process described in Japanese laid-openpatent application 28521/1986 specification.

Polylactic acid for use in the present invention may be whichever of L-and D-compounds and any mixture thereof. Preferred is a species with aD-/L-ratio (mole %) in the range of about 75/25 through about 20180. Themore preferred D-/L-ratio (mole %) of polylactic acid is about 60/40through about 25/75. The most advantageous D/L-ratio (mole %) ofpolylactic acid is about 55/45 through about 25/75. The weight averagemolecular weight of polylactic acid is preferably in the range of about1,500 to about 30,000, more preferably about 2,000 to about 20,000 andstill more preferably about 3,000 to about 15,000. The dispersion valueof polylactic acid is preferably about 1.2 to about 4.0 and moredesirably about 1.5 to about 3.5.

Polylactic acid can be synthesized by two known alternative processes,namely a process involving a ring-opening polymerization of lactidewhich is a dimer of lactic acid and a process involving a dehydrativepolycondensation of lactic acid. For the production of a polylactic acidof comparatively low molecular weight for use in the present invention,the process involving a direct dehydrative polycondensation of lacticacid is preferred. This process is described in, for example, Japaneselaid-open patent application 28521/1986.

In the pharmaceutical base for use in the present invention, theglycolic acid copolymer (A) and polylactic acid (B) are used in an(A)/(B) ratio (by weight) of about 10/90 through about 90/10. Thepreferred blend ratio is about 20/80 through about 80/20. The mostdesirable ratio is about 30/70 through about 70/30. If the proportion ofeither (A) or (B) is too large, the final preparation will show a drugrelease pattern not much different from the pattern obtained when (A) or(B) alone is used, that is to say the linear release pattern in a latestage of release which is obtainable with the mixed base cannot beobtained. The degradation and elimination rates of glycolic acidcopolymer and polylactic acid vary considerably with their molecularweights and composition but generally speaking, since the decompositionand elimination rates of glycolic acid copolymer are relatively higher,the period of release can be prolonged by increasing the molecularweight of polylactic acid or reducing the blend ratio (A)/(B).Conversely, the duration of release may be shortened by reducing themolecular weight of polylactic acid or increasing the (A)/(B) blendratio. Furthermore, the duration of release can be adjusted by changingthe species or relative amount of hydroxycarboxylic acid of generalformula [II].

When a copolymer of lactic acid and glycolic acid is used as thebiodegradable polymer, its polymerization ratio (lactic acid/glycolicacid) (mole %) is preferably about 100/0 to about 40/60. The morepreferred ratio is about 90/10 to about 50/50.

The weight average molecular weight of said copolymer is preferablyabout 5,000 to about 25,000. The more preferred range is about 7,000 toabout 20,000.

The degree of dispersion (weight average molecular weight/number averagemolecular weight) of said copolymer is preferably about 1.2 to about4.0. The more preferred range is about 1.5 to about 3.5.

The above-mentioned copolymer of lactic acid and glycolic acid can besynthesized by the known technology, for example by the processdescribed in Japanese laid-open patent application 28521/1986.

The decomposition and disappearance rate of a copolymer of lactic acidand glycolic acid varies greatly with the composition and molecularweight but generally speaking, the smaller the glycolic acid fraction,the lower is the decomposition and disappearance rate. Therefore, theduration of drug release can be prolonged by reducing the glycolic acidfraction or increasing the molecular weight. Conversely, the duration ofrelease can be diminished by increasing the glycolic acid fraction orreducing the molecular weight. To provide a long-term (e.g. 1˜4 months)sustained-release preparation, it is preferable to use a copolymer oflactic acid and glycolic acid with a polymerization ratio within theabove-mentioned range and a weight average molecular weight within theabove-mentioned range. With a copolymer of lactic acid and glycolic acidhaving a higher decomposition rate than that within the above ranges forpolymerization ratio and weight average molecular weight, it isdifficult to control the initial burst. On the contrary, with acopolymer of lactic acid and glycolic acid showing a lower decompositionrate than that within said ranges for polymerization ratio and weightaverage molecular weight, periods in which the drug will not be releasedin an effective amount tend to occur.

In this specification, the weight average molecular weight and thedegree of dispersion mean the molecular weight in terms of polystyreneas determined by gel permeation chromatography (GPC) using 9polystyrenes with the weight average molecular weights of 120,000,52,000, 22,000, 9,200, 5,050, 2950, 1,050, 580 and 162 as references andthe dispersion value calculated using the same molecular weight,respectively. The above determination was carried out using GPC ColumnKF804 L×2 (Showa Denko), RI Monitor L-3300 (Hitachi) and, as the mobilephase, chloroform.

The sustained-release preparation of the present invention is producedby dissolving the peptide [I] and a biodegradable polymer having aterminal carboxyl group in a solvent which is substantially immisciblewith water and then removing said solvent.

The solvent which is substantially immiscible with water is a solventwhich is not only substantially immiscible with water and capable ofdissolving the biodegradable polymer but one which renders the resultantpolymer solution capable of dissolving the peptide [I]. Preferably, itis a solvent with a solubility in water of not more than 3% (w/w) atatmospheric temperature (20° C.). The boiling point of such solvent ispreferably not higher than 120° C. The solvent, thus, includeshalogenated hydrocarbons (e.g. dichloromethane, chloroform,chloroethane, trichloroethane, carbon tetrachloride, etc.), alkyl ethersof 3 or more carbon atoms (e.g. isopropyl ether etc.), fatty acid alkyl(of 4 or more carbon atoms) esters (e.g. butyl acetate etc.), aromatichydrocarbons (e.g. benzene, toluene, xylene, etc.) and so on. Thesesolvents can be used in a suitable combination of 2 or more species. Themore preferred solvents are halogenated hydrocarbons (e.g.dichloromethane, chloroform, chloroethane, trichloroethane, carbontetrachloride, etc.). The most preferred is dichloromethane.

Removal of the solvent can be effected by the per se known procedures.For example, the method comprising evaporating the solvent atatmospheric pressure or under gradual decompression with constantstirring by means of a propeller mixer or a magnetic stirrer or themethod comprising evaporating the solvent under controlled vacuum in arotary evaporator can be employed.

Referring to the method of the invention for the production of thesustained-release preparation, dissolving the peptide [I] and abiodegradable polymer with a terminal carboxyl group means achieving acondition such that the resultant solution shows no visually observableresidue of undissolved peptide at ordinary temperature (20° C.). In thisternary system consisting of the peptide [I], biodegradable polymer andsolvent, the amount of peptide which can be dissolved depends on thenumber of a terminal carboxyl groups per unit weight of thebiodegradable polymer. In case the peptide and the terminal carboxylgroup interact in the ratio of 1 to 1, the same molar amount of thepeptide as that of the terminal carboxyl group can be dissolved intheory. Therefore, generalization is difficult according to thecombination of the solvent and the molecular weight of the peptide andthe biodegradable polymer. However, in producing sustained-releasepreparations, the peptide may be dissolved in the range of about 0.1 toabout 100% (w/w), preferably about 1 to about 70% (w/w), most preferablyabout 2 to about 50% (w/w), with respect to the biodegradable polymerwhich is dissolved in the solvent.

The present invention is further related to a method of producing asustained-release preparation which comprises dissolving a biodegradablepolymer comprising a mixture of (A) a copolymer of glycolic acid and ahydroxycarboxylic acid of the general formula:

wherein R represents an alkyl group of 2 to 8 carbon atoms and (B) apolylactic acid and a substantially water-insoluble physiologicallyactive peptide or a salt thereof in a solvent which is substantiallyimmiscible with water and then removing said solvent.

The substantially water-insoluble physiologically active peptide is notlimited and includes naturally-occurring, synthetic andsemi-synthetic-peptides. Preferred are physiologically active peptidescontaining one or more aromatic groups (e.g. groups derived frombenzene, naphthalene, phenanthrene, anthracene, pyridine, pyrole,indole, etc.) in side chains thereof. More preferred physiologicallyactive peptides are those having 2 or more aromatic groups in sidechains thereof. Particularly preferred physiologically active peptidesare those having 3 or more aromatic groups in side chains thereof. Thesearomatic groups may be further substituted.

The substantially water-insoluble physiologically active peptide for usein the present invention is preferably a peptide showing a solubility ofnot more than 1% in water, consisting of two or more amino acids andhaving a molecular weight of about 200 to 30000. The molecular weightrange is more preferably about 300 to 20000 and still more preferablyabout 500 to 10000.

As examples of said physiologically active peptide may be mentionedluteinizing hormone releasing hormone (LH-RH) antagonists (cf. U.S. Pat.Nos. 4,086,219, 4,124,577, 4,253,997 and 4,317,815, etc.), in-sulin,somatostatin, somatostatin derivatives (cf. U.S. Pat. Nos. 4,087,390,4,093,574, 4,100,117, 4,253,998, etc.), growth hormone, prolactin,adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone(MSH), salts and derivatives of thyroid hormone releasing hormone (cf.JP Kokai S-50-121273 and S-52-116465), thyroid stimulating hormone(TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH),vasopressin, vasopressin derivatives, oxytocin, calcitonin, gastrin,secretin, pancreozymin, cholecystokinin, angiotensin, human placentallactogen, human chorionic gonadotropin (HCG), enkepharin, enkephalinderivatives (cf. U.S. Pat. No. 4,277,394, EP-A No. 31,567), endorphin,kyotrphin, tuftsin, thymopoietin, thymosin, thymostimulin, thymichumoral factor (THF), facteur thymique serique (FTS) and its derivatives(cf. U.S. Pat. No. 4,229,438), other thymic factors, tumor necrosisfactor (TNF), colony stimulating factor (CSF), motilin, dynorphin,bombesin, neurotensin, cerulein, bradykinin, atrial natruretic factor,nerve growth factor, cell growth factor, neurotrophic factor, peptideshaving endothelin antagonistic activity (cf. EP-A No. 436189, No. 457195and No. 496452, JP Kokai H-3-94692 and 03-130299) and fragments orderivatives of these physiologically active peptides.

Specific examples of the physiologically active peptide arephysiologically active peptides and salts which are antagonists ofluteinizing hormone releasing hormone (LH-RH) and useful for thetreatment of hormone-dependent diseases such as prostatic cancer,prostatic hypertrophy, endometriosis, uterine myoma, precocious puberty,breast cancer, etc. and for contraception.

The physiologically active peptide for use in the present invention canbe in the form of a salt, preferably a pharmacologically acceptablesalt. Where said peptide has a basic group such as amino, the saltmentioned above may for example be the salt formed with an inorganicacid (e.g. hydrochloric acid, sulfuric acid, nitric acid, etc.) or anorganic acid (e.g. carbonic acid, hydrogencarbonic acid, succinic acid,acetic acid, propionic acid, trifluoroacetic acid, etc.). Where thepeptide has an acidic group such as carboxyl, the salt may for examplebe the salt formed with an inorganic base (e.g. alkali metals such assodium, potassium, etc. and alkaline earth metals such as calcium,magnesium, etc.) or an organic base (e.g. organic amines such astriethylamine etc. and basic amino acids such as arginine etc.). Thepeptide may further be in the form of a metal complex compound (e.g.copper complex, zinc complex, etc.).

Specific examples of the physiologically active peptide or salt thereofare found in U.S. Pat. No. 5,110,904, Journal of Medicinal Chemistry 34,2395-2402, 1991, Recent Results in Cancer Research 124, 113-136, 1992,and other literature.

Furthermore, the physiologically active peptides of general formula [I]and salts thereof can also be mentioned, among others.

Moreover, even when the physiologically active peptide is water-soluble,it can be converted to a derivative compound which is insoluble orconverted to an insoluble salt with a water-insoluble acid (e.g. pamoicacid, tannic acid, stearic acid, palmitic acid, etc.) and used in theprocess of the invention.

The amount of said physiologically active peptide in the preparations ofthe present invention depends on the species of peptide, expectedpharmacologic effect and desired duration of effect and so on.Generally, however, it is used in a proportion of about 0.001 to 50%(w/w), preferably about 0.01 to 40% (w/w), more preferably about 0.1 to30% (w/w), relative to the biodegradable polymer base.

The solvent employed in the method is the same as described above.

Removal of the solvent can be carried out in the same manner asdescribed above.

The preferred process for the production of the sustaietd-releasepreparation of the present invention is a microencapsulating processutilizing the drying-in-water technique or the phase separationtechnique, which is described below, or any process analogous thereto.

The process described below may be carried out with peptide [I] or witha substantially water-insoluble physiologically active peptide whichincludes peptide [I].

Thus, the peptide [I] is added to a solution of the biodegradablepolymer in an organic solvent in the final weight ratio mentionedhereinbefore for such peptide to prepare an organic solvent solutioncontaining the peptide [I] and biodegradable polymer. In thisconnection, the concentration of the biodegradable polymer in theorganic solvent varies according to the molecular weight of thebiodegradable polymer and the type of organic solvent but is generallyselected from the range of about 0.01 to about 80% (w/w). The preferredrange is about 0.1 to about 70% (w/w). The still more preferred range isabout 1 to about 60% (w/w).

Then, this organic solvent solution containing the peptide [I] andbiodegradable polymer (oil phase) is added to a water phase to preparean O(oil phase)/W (water phase) emulsion. The solvent of the oil phaseis then evaporated off to provide microcapsules. The volume of the waterphase for this procedure is generally selected from the range of about 1to about 10000 times the volume of the oil phase. The preferred range isabout 2 to about 5000 times and the still more preferred range is about5 to about 2000 times.

An emulsifier may be added to the above water phase. The emulsifier maygenerally be any substance that contributes to the formation of a stableO/W emulsion. Thus, there can be mentioned anionic surfaet-ants (sodiumoleate, sodium stearate, sodium lauryl sulfate, etc.), nonionicsurfactants (polyoxyethylene-sorbitan fatty acid esters [Tween 80 andTween 60, Atlas Powder], polyoxyethylene-castor oil derivatives [HCO-60and HCO-50, Nikko Chemicals], etc.), polyvinylpyrrolidone, polyvinylalcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid andso on. These emulsifiers can be used independently or in combination.The concentration may be selected from the range of about 0.001 to about20% (w/w). The preferred range is about 0.01 to about 10% (w/w) and thestill more preferred range is about 0.05 to about 5% (w/w).

The resultant microcapsules are recovered by centrifugation orfiltration and washed with several portions of distilled water to removethe free peptide, vehicle and emulsifier from the surface, thenredispersed in distilled water or the like and lyophilized. Then, ifnecessary, the microcapsules are heated under reduced pressure tofurther remove the residual water and organic solvent from within themicrocapsules. Preferably, this procedure is carried out by heating themicrocapsule at a temperature somewhat (5° C. or more) above the medianglass transition temperature of the biodegradable polymer as determinedwith a differential scanning calorimeter at temperature increments of 10to 20° C./min., generally for not more than 1 week or 2 to 3 days,preferably for not more than 24 hours, after the microcapsules havereached the target temperature.

In the production of microcapsules by the phase separation technique, acoacervation agent is gradually added to a solution of said peptide [I]and biodegradable polymer in an organic solvent with constant stirringso that the biodegradable polymer may separate out and solidify. Thiscoacervation agent is added in a volume of about 0.01 to about 1000times the volume of the organic solvent solution of peptide [I] andbiodegradable polymer. The preferred range is about 0.05 to about 500times and the still more preferred range is about 0.1 to about 200times.

The coacervation agent should be a compound of polymer, mineral oil orvegetable oil type which is miscible with the solvent for thebiodegradable polymer yet which does not dissolve the polymer.Specifically, silicone oil, sesame oil, soybean oil, corn oil,cottonseed oil, coconut oil, linseed oil, mineral oil, n-hexane,n-heptane, etc. can be mentioned. These substances can be used incombination.

The resultant microcapsules are recovered by filtration and washedrepeatedly with heptane or the like to remove the coacervation agent.Then, the free peptide and solvent are removed by the same procedure asdescribed for the drying-in-water technique.

In the drying-in-water technique or in the coacervation technique, anaggregation inhibitor may be added so as to prevent aggregation ofparticles. The aggregation inhibitor includes water-solublepolysaccharides such as mannitol, lactose, glucose, starch (e.g. cornstarch), etc., glycine, proteins such as fibrin, collagen, etc., andinorganic salts such as sodium chloride, sodium hydrogen phosphate andso on.

In the production of microcapsules by the spray drying technique, saidorganic solvent solution of peptide [I] and biodegradable polymer isejected in a mist form through a nozzle into the drying chamber of aspray drier to evaporate the organic solvent from the finely-dividedliquid droplets in a brief time to provide fine microcapsules. Thenozzle may be a two-fluid nozzle, pressure nozzle, rotary disk nozzleand so on. It is advantageous to the process to spray an aquaoussolution of said aggregation inhibitor from another nozzle for theprevention of intercapsule aggregation in timed coordination with saidspray of the organic solvent solution of peptide [I] and biodegradablepolymer.

If necessary, the residual water and organic solvent are removed byheating the resultant microcapsules under reduced pressure in the samemanner as described hereinbefore.

The microcapsules can be administered as they are or as processed intovarious pharmaceutical preparations for administration by routes otherthan peroral (e.g. intramuscular, subcutaneous and intraorgan injectionsor implants, nasal, rectal or uterine transmucosal delivery systems, andso on) or for oral administration (e.g. solid preparations such ascapsules (e.g. hard capsules, soft capsules, etc.), granules, powders,etc. and liquid preparations such as syrups, emulsions, suspensions andso on).

To process the microcapsules for injection, for instance, themicrocapsules can be formulated with a dispersant (e.g. a surfactantsuch as Tween 80, HCO-60, etc., carboxymethylcellulose, a polysaccharidesuch as sodium alginate, etc.), a preservative (e.g. methylparaben,propylparaben, etc.), or an isotonizing agent (e.g. sodium chloride,mannitol, sorbitol, glucose, etc.) to prepare an aqueous suspension orthey may be dispersed in a vegetable oil such as sesame oil, corn oil orthe like to provide an oil suspension for use as a controlled releaseinjection.

The particle size of the microcapsules for such injectable suspensionsneed only be in the range satisfying the dispersibility and needlepassage requirements and may for example range from about 0.1 to about500 μm. The preferred particle size range is about 1 to about 300 μm andthe still more preferred range is about 2 to about 200 μm.

For providing the microcapsules as a sterile product, the wholeproduction process is subjected to sterility control, the microcapsulesare sterilized by gamma-ray irradiation or a preservative is added,although these are not exclusive procedures.

Aside from the above-mentioned microcapsules, a biodegradable polymercomposition containing the active ingredient peptide well dispersed by asuitable technique can be melted and molded into a spherical,bar-shaped, needle-shaped, pelletized or film shape to provide asustained-release preparation of the present invention. The abovebiodegradable polymer composition can be produced by the methoddescribed in JP Publication S-50-17525. To be specific, the peptide drugand the polymer are dissolved in a solvent and the solvent is thenremoved by a suitable method (e.g. spray drying, flash evaporation,etc.) to provide the desired biodegradable polymer composition.

The sustained-release preparation of the present invention can beadministered as an intramuscular, subcutaneous or intraorgan injectionor implant, a transmucosal delivery system for application to the nasalcavity, rectum or uterus, or an oral preparation (e.g. a solidpreparation such as a capsule (e.g. hard or soft), granule, powder, etc.or a liquid preparation such as syrup, emulsion, suspension, etc.).

The sustained-release preparation of the present invention has lowtoxicity and can be used safely in mammalian animals (e.g. man, bovine,swine, canine, feline, murine, rat and rabbit).

The dosage of the sustained-release preparation is dependent on the typeand content of the active drug peptide, final dosage form, the durationof release of the peptide, the object of treatment (such ashormone-dependent diseases, e.g. prostatic cancer, prostatomegaly,endometriosis, metrofibroma, precocious puberty, mammary cancer, etc.,or for contraception) and the subject animal species, but in any case itis necessary that an effective amount of peptide is successfullydelivered. The unit dosage of the active drug peptide, taking aone-month delivery system as an example, can be selected advantageouslyfrom the range of about 0.01 to about 100 mg/kg body weight for an adulthuman. The preferred range is about 0.05 to about 50 mg/kg body weight.The most preferred range is about 0.1 to about 10 mg/kg body weight.

The unit dosage of the sustained-release preparation per adult human cantherefore be selected from the range of about 0.1 to about 500 mg/kgbody weight. The preferred range is about 0.2 to about 300 mg/kg bodyweight. The frequency of administration may range from once in a fewweeks, monthly or once in a few months, for instance, and can beselected according to the type and content of the active drug peptide,final dosage form, designed duration of release of the peptide, thedisease to be managed and the subject animal.

The following reference and working examples are intended to describethe invention in further detail and should by no means be construed asdefining the scope of the invention. (Unless otherwise specified, %means % by weight).

Abbreviations used hereinafter have the following definitions:

BOC: tert-butoxycarbonyl

FMOC: 9-fluorenylmethoxycarbonyl

Cbz: Benzyloxycarbonyl

Reference Example 1

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 300 g of 90% aqueous solution of D,L-lacticacid and 100 g of 90.-queous solution of L-lactic acid and the chargewas heated under reduced pressure in a nitrogen gas stream from 100°C./500 mmHg to 150° C./30 mmHg over a period of 4 hours, with thedistillate water being constantly removed. The reaction mixture wasfurther heated under reduced pressure at 3-5 mmHg/150-180° C. for 7hours, after which it was cooled to provide an amber-colored polylacticacid.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the above polylactic acid were 3,000;1,790; and 1,297, respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 2

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 500 g of 90% aqueous solution of D,L-lacticacid and the charge was heated under reduced pressure in a nitrogen gasstream from 100° C./500 mmHg to 150° C./30 mmHg for a period of 4 hours,with the distillate water being constantly removed. The reaction mixturewas further heated under reduced pressure at 3-5 mmHg/150-180° C. for 12hours, after which it was cooled to provide an amber-colored polylacticacid.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the above polylactic acid was 5,000;2,561; and 1,830, respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 3

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 300 g of 90% aqueous solution of D,L-lacticacid and 100 g of 90% aqueous solution of L-lactic acid and the chargewas heated under reduced pressure in a nitrogen gas stream from 100°C./500 mmHg to 150° C./30 mmHg for a period of 5 hours, with thedistillate water being constantly removed. The reaction mixture wasfurther heated under reduced pressure at 5-7 mmHg/150-180° C. for 18hours, after which it was cooled to provide an amber-colored polylacticacid.

This polymer was dissolved in 1000 ml of dichloro-methane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the above polylactic acid was 7,500,3,563; and 2,301, respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 4

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 300 g of 90% aqueous solution of D,L-lacticacid and 100 g of 90% aqueous solution of L-lactic acid and the chargewas-heated under reduced pressure in a nitrogen gas stream from 100°C./500 mmHg to 150° C./30 mmHg for a period of 5 hours, with thedistillate water being constantly removed. The reaction mixture wasfurther heated under reduced pressure at 5-7 mmHg/150-180° C. for 26hours, after which it was cooled to provide an amber-colored polylacticacid.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the above polylactic acid was 9,000;3,803; and 2,800, respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 5

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 182.5 g of glycolic acid and 166.6 g ofD,L-2-hydroxybutyric acid and the charge was heated under reducedpressure in a nitrogen gas stream from 100° C./500 mmHg to 150° C./30mmHg for a period of 3.5 hours, with the distillate water beingconstantly removed. The reaction mixture was further heated underreduced pressure at 5-7 mmHg/150-180° C. for 26 hours, after which itwas cooled to provide an amber-colored glycolic acid-2-hydroxybutyricacid copolymer.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 25° C.

The weight average molecular weight, as determined by GPC, of theresulting glycolic acid-2-hydroxybutyric acid copolymer was 13,000.

Reference Example 6

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondensor was charged with 197.7 g of glycolic acid and 145.8 g ofD,L-2-hydroxybutyric acid and the charge was heated under reducedpressure in a nitrogen gas stream from 100° C./500 mmHg to 155° C./30mmHg for a period of 4 hours, with the distillate water being constantlyremoved. The reaction mixture was further heated under reduced pressureat 3-6 mmHg/150-185° C. for 27 hours, after which it was cooled toprovide an amber-colored glycolic acid-2-hydroxybutyric acid copolymer.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 25° C.

The weight average molecular weight, as determined by GPC, of theresulting glycolic acid-2-hydroxybutyric acid copolymer was 13,000.

Reference Example 7

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 212.9 g of glycolic acid and 124.9 g ofD,L-2-hydroxybutyric acid and the charge was heated under reducedpressure in a nitrogen gas stream from 100° C./500 mmHg to 160° C./30mmHg for a period of 3.5 hours, with the distillate water beingconstantly removed. The reaction mixture was further heated underreduced pressure at 3-6 mmHg/160-180° C. for 27 hours, after which itwas cooled to provide an amber-colored glycolic acid-2-hydroxybutyricacid copolymer.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 25° C.

The weight average molecular weight, as determined by GPC, of theresulting glycolic acid-2-hydroxybutyric acid copolymer was 11,000.

Reference Example 8

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondensor was charged with 300 g of 90% aqueous solution of D,L-lacticacid and 100 g of 90% aqueous solution of L-lactic acid and the chargewas heated under reduced pressure in a nitrogen gas stream from 100°C./500 mmHg to 150° C./30 mmHg for a period of 4 hour with thedistillate water being constantly removed. The reaction mixture wasfurther heated under reduced pressure at 3-5 mmHg and 150-180° C. for 10hours, after which it was cooled to provide an amber-colored polylacticacid.

This polymer was dissolved in 1,000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight-average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the above polylactic acid was 4,200;2,192; and 1,572, respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 9

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 182.5 g of glycolic acid and 166.6 g ofD,L-2-hydroxybutyric acid and the charge was heated under reducedpressure in a nitrogen gas stream from 100° C./500 mmHg to 150° C./30mmHg for a period of 3.5-hour, with the distillate water beingconstantly removed. The reaction mixture was further heated underreduced pressure at 5-7 mmHg and 150-180° C. for 32 hours, after whichit was cooled to provide an amber-colored glycolic acid-2-hydroxybutyricacid copolymer.

The polymer was dissolved in 1,000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in avacuo at 25° C.

The weight-average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the resulting glycolicacid·2-hydroxybutyric acid copolymer was 16,300; 5,620; and 2,904,respectively.

These data showed that the polymer had terminal carboxyl groups.

Reference Example 10

Synthesis ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂

Reference Examples 10 and 11 were carried out in accordance with thedescription of U.S. Pat. No. 5,110,904 and U.S. patent application No.07/987,921.

To the reactor of a peptide synthesizer was added 1 g of D-Ala-NH-resin(4-methyl-benzohydrylamine resin), followed by sequential additions ofamino acids per the following synthesis procedure, to synthesize thetitle peptide.

1. Deprotecting reaction

To remove the protecting BOC group from the peptide's alpha amino acid,a solution consisting of 45% trifluoroacetic acid (hereinafter alsoreferred to as TFA), 2.5% anisole, 2.0% dimethyl phosphite and 50.5%dichloromethane was used. After the resin was pre-washed with thesolution for 1 minute, a deprotecting reaction was conducted for 20minutes.

2. Washing with basic solution

To remove and neutralize the trifluoroacetic acid used for deprotection,a dichloromethane solution containing 10% N,N′-diisopropylethylamine wasused. The resin was washed for 1 minute three times for eachdeprotecting reaction.

3. Coupling reaction

A coupling reaction was carried out, using as activators a 3-fold molaramount of 0.3 M diisopropylcarbodiimide/dichloromethane solution and a3-fold molar amount of 0.3 M BOC amino acid derivative/DMF(N,N′-dimethylformamide) solution. The activated amino acid was coupledto the free alpha amino group of the peptide on the resin. Reactiontimes are shown below.

4. Washing

After completion of every reaction process, the resin was washed withdichloromethane, dichloromethane/DMF and DMF, each for 1 minute.

Synthesis protocol

Amino-group-protected amino acids were coupled to the resin in theorder, frequency and time shown below.

Order Amino acid Frequency time 1 BOC-Pro 2 times 1 hour 2BOC-Lys(N-epsilon- 2 times 1 hour Cbz, isopropyl) 3 BOC-Leu 2 times 1hour 4 BOC-D-Lys 2 times 1 hour (N-epsilon-FMOC) 5 BOC-NMeTyr 2 times 1hour (O-2,6-diCl-Bzl) 6 BOC-Ser(OBzl) 2 times 1 hour 7 BOC-D-3Pal 2times 6 hours 8 BOC-D-4ClPhe 2 times 2 hours 9 BOC-D2Nal 2 times 2 hours10 Acetic acid 2 times 2 hours

After completion of the synthesis reaction, the resin was treated with a30% piperidine solution in DMF for 4 to 24 hours to remove theprotecting FMOC group. The resin was washed with dichloromethane severaltimes and then reacted with carbonyldiimidazole (0.9 g) dissolved in DMF(18 ml) for 15 minutes and washed with dichloromethane three times,after which it was reacted overnight with 2-furoic hydrazide (0.53 g)dissolved in DMF (18 ml). The resulting peptide-resin was washed withdichloromethane three times and then dried in the presence of phosphoruspentoxide overnight, after which it was treated with dry hydrogenfluoride at 0° C. for 1 hour in the presence of anisole to cut thepeptide from the resin. The excess reaction reagent was removed undervacuum conditions. The thus-obtained resin was first washed with ether,then stirred at room temperature for 15 minutes in 50 ml of awater/acetonitrile/acetic acid mixture (1:1:0.1) and filtered. Thefiltrate was lyophilized to yield an unpurified peptide as a fluffypowder. This peptide was purified by high performance liquidchromatography (HPLC) under the following conditions.

(1) Column: Dynamax C-18 (25×2.5 cm, 8 microns)

(2) Solvent: Acetonitrile ascending gradient over a 20-minute periodfrom 89% water/11% acetonitrile/0.1% TFA

(3) Detection wavelength: 260 nm (UV method)

The peptide detected as a single peak at 25.7 minutes retention time wascollected and lyophilized to yield a purified product ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂as a trifluoroacetate. Physical property data on the purified productare as follows:

FAB (fast atom bombardment, the same applies below) mass spectrometry:m/e 1591 (M+H)⁺

Amino acid analysis: 0.98 Ala, 1.02 Pro, 1.58 Lys, 1.00 Leu, 1.12NMeTyr, 0.52 Ser

The above trifluoroacetate of peptide was converted to an acetate, usinga gel filtration column previously equilibrated with 1 N acetic acid.Gel filtration conditions are as follows:

(1) Packing: Sephadex G-25 (column inside diameter 16 mm, packing bedheight 40 mm)

(2) Solvent: 1 N acetic acid

(3) Detection wavelength: 254 nm (UV method)

The fraction of the first eluted peak was collected and lyophilized toyield a purified product ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂as an acetate.

Reference Example 11

Synthesis ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH₂

The title peptide was synthesized in the same manner as in ReferenceExample 10, except that 2-furoic hydrazide was replaced with 2-nicotinichydrazide (0.575 g). The HPLC retention time of the purified productthus obtained was 16.0 minutes. Physical property data on the purifiedproduct are as follows:

FAB mass spectrometry: m/e 1592 (M+H)⁺

Amino acid analysis: 1.02 Ala, 1.01 Pro, 1.61 Lys, 0.99 Leu, 1.12NMeTyr, 0.48 Ser

The above trifluoroacetate of peptide was converted to an acetate in thesame manner as in Reference Example 10.

Reference Example 12

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 322 g of 90% aqueous solution of D,L-lacticacid and 133 g of glycolic acid and using a mantle heater (So-goRikagaku Glass Co.), the charge was heated under reduced pressure in anitrogen stream from 100° C./500 mmHg to 150° C./30 mmHg for a period of4 hours the distillate water being constantly removed. The reactionmixture was further heated under reduced pressure at 3-30 mmHg/150-185°C. for 23 hours, after which it was cooled to provide a lacticacid-glycolic acid copolymer.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the resultant lactic acid-glycolic acidcopolymer were 10,000; 4,000; and 4,000, respectively. These data showedthat the copolymer was a polymer having terminal carboxyl groups.

Reference Example 13

A 1000 ml four-necked flask equipped with a nitrogen inlet pipe andcondenser was charged with 347 g of 90% aqueous solution of D,L-lacticacid and 266 g of glycolic acid and using a mantle heater (So-goRikagaku Glass Co.), the charge was heated under reduced pressure in anitrogen stream from 100° C./500 mmHg to 150° C./30 mmHg for a period of5 hours, with the distillate water being constantly removed. Thereaction mixture was further heated under reduced pressure at 3-30mmHg/150-185° C. for 23 hours, after which it was cooled to provide alactic acid-glycolic acid copolymer.

This polymer was dissolved in 1000 ml of dichloromethane and thesolution was poured in warm water at 60° C. with constant stirring. Theresulting pasty polymeric precipitates were collected and dried in vacuoat 30° C.

The weight average molecular weight and number average molecular weight,as determined by GPC, and the number average molecular weight, as foundby end-group determination, of the resultant lactic acid-glycolic acidcopolymer were 10,000; 3,700; and 3,900, respectively. These data showedthat the copolymer was a polymer having terminal carboxyl groups.

EXAMPLE 1

NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂(manufactured by TAP; hereinafter referred to briefly as physiologicallyactive peptide A) acetate, 200 mg, was dissolved in a solution of a50:50 mixture (3.8 g) of the glycolic acid-2-hydroxybutyric acidcopolymer obtained in Reference Example 5 and the polylactic acidobtained in Reference Example 1 in 5.3 g (4.0 ml) of dichloromethane.The resulting solution was cooled to 17° C. and poured into 1000 ml of a0.1% (w/w) aqueous solution of polyvinyl alcohol (EG-40, NipponSynthetic Chemical Industry Co., Ltd.) previously adjusted to 10° C. andthe mixture was emulsified using a turbine homomixer at 7000 rpm toprepare an O/W emulsion. This O/W emulsion was stirred at roomtemperature for 3 hours to evaporate the dichloromethane. The oil phasewas solidified and collected with a centrifuge (05PR-22, Hitachi, Ltd.)at 2000 rpm. This solid was redispersed in distilled water and furthercentrifuged to wash off the free drug etc. The collected microcapsuleswere redispersed in a small quantity of distilled water, followed byaddition of 0.3 g of D-mannitol and freeze-drying to provide a powder.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 5 to 60 μm and 4.7% (w/w),respectively.

Preparations of the following physiologically active peptides (1) and(2) were manufactured in the same manner as above.

(1)NAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH₂

(2)NAcD2Nal-D4ClPhe-D3Pal-ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂

EXAMPLE 2

In a solution of a 50:50 mixture (3.8 g) of the glycolicacid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 andthe polylactic acid obtained in Reference Example 2 in 6.7 g (5.0 ml) ofdichloromethane was dissolved 200 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17° C.and the mixture was treated as in Example 1 to provide microcapsules.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 5 to 65 μm and 5.0% (w/w),respectively.

EXAMPLE 3

In a solution of a 50:50 mixture (3.8 g) of the glycolicacid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 andthe polylactic acid obtained in Reference Example 3 in 6.7 g (5.0 ml) ofdichloromethane was dissolved 200 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17° C.and the mixture was treated as in Example 1 to provide microcapsules.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 10 to 60 μm and 4.8% (w/w),respectively.

EXAMPLE 4

In a solution of a 50:50 mixture (3.8 g) of the glycolicacid-2-hydroxybutyric acid copolymer obtained in Reference Example 5 andthe polylactic acid obtained in Reference Example 4 in 6.7 g (5.0 ml) ofdichloromethane was dissolved 200 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17° C.and the mixture was treated as in Example 1 to provide microcapsules.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 10 to 75 μm and 4.6% (w/w),respectively.

EXAMPLE 5

In a solution of a 50:50 mixture (3.8 g) of the glycolicacid-2-hydroxybutyric acid copolymer obtained in Reference Example 6 andthe polylactic acid obtained in Reference Example 2 in 6.0 g (4.5 ml) ofdichloromethane was dissolved 200 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1000 ml of a0.1% aqueous solution Hf polyvinyl alcohol previously adjusted to 10° C.and the mixture was treated as in Example 1 to provide microcapsules.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 5 to 60 μm and 4.9% (w/w),respectively.

EXAMPLE 6

In a solution of a 50:50 mixture (3.8 g) of the glycolicacid-2-hydroxybutyric acid copolymer obtained in Reference Example 7 andthe polylactic acid obtained Reference Example 2 in 6.0 g (4.5 ml) ofdichloromethane was dissolved 200 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17° C.and the mixture was treated as in Example 1 to provide microcapsules.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 10 to 65 μm and 4.9% (w/w),respectively.

EXAMPLE 7

In a solution of a 50:50 mixture (3.6 g) of the glycolicacid.2-hydroxybutyric acid copolymer obtained in Reference Example 9 andthe polylactic acid obtained in Reference Example 8 in 7.0 g (5.3 ml) ofdichloromethane was dissolved 400 mg of physiologically active peptide Aacetate. This solution was cooled to 17° C. and poured into 1,000 ml ofa 0.1% aqueous solution of polyvinyl alcohol previously adjusted to 17°C. and the mixture was treated as in Example 1, to providemicrocapsules. The particle size distribution and physiologically activepeptide A content of the microcapsules were 5 to 65 μm and 7.2% (w/w),respectively.

EXAMPLE 8

240 mg of the acetate ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyNic)-Leu-Lys(Nisp)-Pro-DAlaNH₂(hereinafter referred to briefly as physiologically active peptide B)obtained in Reference Example 11 was dissolved in a solution of a 50:50mixture (1.76 g) of the glycolic acid.2-hydroxybutyric acid copolymerobtained in Reference Example 9 and the polylactic acid obtained inReference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resultingsolution was cooled to 18° C. and poured into 400 ml of a 0.1% aqueoussolution of polyvinyl alcohol previously adjusted to 16° C. and themixture was treated as in Example 1, to provide microcapsules. Theparticle size distribution and physiologically active peptide B contentof the microcapsules were 5 to 70 μm and 10.3% (w/w), respectively.

EXAMPLE 9

240 mg of the acetate ofNAcD2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-DLys(AzaglyFur)-Leu-Lys(Nisp)-Pro-DAlaNH₂(hereinafter referred to briefly as physiologically active peptide C)obtained in Reference Example 10 was dissolved in a solution of a 50:50mixture (1.76 g) of the glycolic acid.2-hydroxybutyric acid copolymerobtained in Reference Example 9 and the polylactic acid obtained inReference Example 8 in 3.2 g (2.4 ml) of dichloromethane. The resultingsolution was cooled to 18° C. and poured into 400 ml of a 0.1% aqueoussolution of polyvinyl alcohol previously adjusted to 16° C. and themixture was treated as in Example 1, to provide microcapsules. Theparticle size distribution and physiologically active peptide C contentof the microcapsules were 5 to 65 μm and 10.9% (w/w), respectively.

EXAMPLE 10

N-Tetrahydrofur-2-oyl-Gly-D2Nal-D4ClPhe-D3Pal-Ser-NMeTyr-Dlys(Nic)-Leu-Lys(Nisp)-Pro-DAlaNH₂(Manufactured by TAP; hereinafter referred to briefly as physiologicallyactivepeptide D) acetate [FAB mass spectrometry: m/e 1647 (M+H)⁺], 240mg, was dissolved in a solution of a 50:50 mixture (1.76 g) of theglycolic acid-2-hydroxybutyric acid copolymer obtained in ReferenceExample 9 and the polylactic acid obtained in Reference Example 8 in 3.2g (2.4 ml) of dichloromethane. The resulting solution was cooled to 18°C. and poured into 400 ml of a 0.1% aqueous solution of polyvinylalcohol previously adjusted to 16° C. and the mixture was treated as inExample 1 to provide microcapsules. The particle size distribution andphysiologically active peptide D content of the microctapsules were 5 to70 μm and 10.5% (w/w), respectively.

EXAMPLE 11

200 mg of physiologically active peptide A acetate was added anddissolved in a solution of a lactic acid-glycolic acid copolymer (lacticacid/glycolic acid=75/25 (mole %), GPC weight average mol. wt.=5,000,GPC number average mol. wt.=2,000, number average mol. wt. by end-groupdetermination=2,200; manufacturer; Wako Pure Chemical (Lot. 920729)) in5.3 g (4.0 ml) of dichloromethane. The resulting solution was cooled to17° C. and poured into 1000 ml of a 0.1% aqueous solution of polyvinylalcohol (EG-40, Nippon Synthetic Chemical Industry Co., Ltd.) previouslyadjusted to 16° C. and the mixture was emulsified using a turbine mixerat 7000 rpm to prepare an O/W emulsion. This O/W emulsion was stirred atroom temperature for 3 hours to evaporate the dichloromethane. The oilphase was solidified and collected with a centrifuge (05PR-22, Hitachi)at 2000 rpm. This solid was redispersed in distilled water and furthercentrifuged to wash off the free drug etc. The collected microcapsuleswere redispersed in a small quantity of distilled water, followed byaddition of 0.3 g of D-mannitol and freeze-drying to provide a powder.The particle size distribution and physiologically active peptide Acontent of the microcapsules were 5 to 60 μm and 4.7% (w/w),respectively.

Sustained-release preparation of the following peptides (1) and (2) areproduced in the same manner as above.

(1) Physiologically active peptide B acetate

(2) Physiologically active peptide C acetate

EXAMPLE 12

200 mg of physiologically active peptide A acetate was added anddissolved in a solution of 3.8 g of a lactscadcid-glycolic copolymer(lactic acid/glycolic acid=75/25 (mole %), GPC weight average mol.wt.=10,000, GPC number average mol. wt.=4,400, number average mol. wt.by end-group determination=4,300;

manufacturer; Wako Pure Chemical (Lot. 880530)) in 6.7 g (5.0 ml) ofdichloromethane. The resulting solution was cooled to 17° C. and pouredinto 1000 ml of a 0.1% aqueous solution of polyvinyl alcohol previouslyadjusted to 11° C. Thereafter, the procedure of Example 11 was repeatedto provide microcapsules. The particle size distribution andphysiologically active peptide A content of the microcapsules were 5 to65 μm and 4.5% (wiw), respectively.

EXAMPLE 13

400 mg of physiologically active peptide A acetate was dissolved in asolution of the lactic acid-glycolic acid copolymer obtained inReference Example 12, 3.6 g, in 8.0 g (6.0 ml) of dichloromethane. Theresulting solution was cooled to 15° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 14° C.Thereafter, the procedure of Example 11 was repeated to providemicrocapsules. The particle size distribution and physiologically activepeptide A content of the microcapsules were 5 to 65 μm and 8.2% (w/w),respectively.

EXAMPLE 14

400 mg of physiologically active peptide A acetate was dissolved in asolution of the lactic acid-glycolic acid copolymer obtained inReference Example 13, 3.6 g, in 8.0 g (6.0 ml) of dichloromethane. Theresulting solution was cooled to 15° C. and poured into 1000 ml of a0.1% aqueous solution of polyvinyl alcohol previously adjusted to 15° C.Thereafter, the procedure of Example 11 was repeated to providemicrocapsules. The particle size distribution and physiologically activepeptide A content of the microcapsules were 5 to 65 μm and 8.4% (w/w),respectively.

EXAMPLE 15

Leuprorelin acetate (Leuprolide, Merck Index 5484, p. 932 (12th ed.1996; manufacturer Takeda Chemical Industries), 400 mg, was added to asolution of the same lactic acid-glycolic acid copolymer as used inExample 12, 3.6 g, in 8.0 g (60 ml) of dichloromethane to prepare aclear homogeneous solution. The resulting solution was cooled to 15° C.and poured into 1000 ml of a 0.1% aqueous solution of polyvinyl alcoholpreviously adjusted to 15° C. Thereafter, the procedure of Example 11was repeated to provide microcapsules.

Experimental Example 1

About 30 mg of the microcapsules obtained in Example 1 were dispersed ina dispersion medium (a solution of 2.5 mg of carboxymethylcellulose, 0.5mg of polysorbate 80 and 25 mg of mannitol in distilled water) and thedispersion was injected subcutaneously in the back of 10-week-old maleSD rats using a 22G needle (the dosage of microcapsules was 60 mg/kg).Serially after administration, the rats were sacrificed, the remnants ofmicrocapsules were taken out from the administration site and the amountof the physiologically active peptide A in the microcapsules wasdetermined. The results are shown in Table 1.

Experimental Examples 2-6

Using the microcapsules obtained in Examples 2 to 6, the residualamounts of the physiologically active peptide A in the microcapsuleswere determined as in Experimental Example 1. The results are also shownin Table 1.

TABLE 1 Residue of physiologically active peptide A (%) Day Week WeekWeek Week Week Week Week 1 1 2 3 4 5 6 8 Experimental 88.0 66.5 42.315.2 Example 1 Experimental 92.8 76.6 62.6 48.7 38.6 26.5 Example 2Experimental 96.5 90.5 77.5 64.9 59.2 46.9 38.7 20.3 Example 3Experimental 99.4 94.5 87.2 76.3 66.0 — 46.6 30.7 Example 4 Experimental92.9 75.0 45.7 — 17.5 Example 5 Experimental 92.3 61.3 33.5  6.4 Example6

It is apparent from Table 1 that all the micro-capsules according to thepresent invention are characterized by substantially constant release ofphysiologically active peptide and are further characterized by beingsubstantially free from an initial burst.

Table 2 shows the linear regression models, correlation coefficients,and release periods calculated as X-intercept which were determined bythe procedures described in Methods of Bioassay (authored by AkiraSakuma, Tokyo University Press, Jun. 5, 1978, p. 111).

TABLE 2 Weight average molecular Linear Release weight of regressionCorrelation period polylactic acid model coefficient (weeks)Experimental 3000 Residue (%) = (R² = 3.5 Example 1  95.4−(26.9 × 0.992)no. of weeks) Experimental 5000 Residue (%) = (R² = 6.6 Example 2 94.4−(14.2 × 0.975) no. of weeks) Experimental 7500 Residue (%) = (R² =9.8 Example 3  98.4−(10.0 × 0.996) no. of weeks) Experimental 9000Residue (%) = (R² = 11.5  Example 4 102.1−(8.9 × 0.995) no. of weeks)

It is apparent from Table 2 that by varying the weight average molecularweight of polylactic acid to be blended with glycolicacid-2-hydroxybutyric copolymer, the duration of release can be freelycontrolled within the range of about 3.5 weeks to about 11.5 weeks.

Table 3 shows the linear regression models, correlation coefficients andrelease periods as X-intercept which were determined from the data inTable 1 by the same procedures as used in Table 2.

TABLE 3 Mole fraction of Cor- Release glycolic acid in relation periodglycolic acid copolymer Linear regression model coefficient (weeks)Experimental 60% Residue (%) = (R² = 6.6 Example 2 94.4−(14.2 × no. of0.975) weeks) Experimental 65% Residue (%) = (R² = 4.6 Example 595.7−(20.6 × no. of 0.976) weeks) Experimental 70% Residue (%) = (R² =3.1 Example 6 96.6−(30.9 × no. of 0.994) weeks)

It is apparent from Table 3 that by varying the mole fraction ofglycolic acid in the glycolic acid-2-hydroxybutyric acid copolymer to beblended with polylactic acid, the duration of release can be freelycontrolled within the range of about 3.1 weeks to about 6.6 weeks.

Experimental Examples 7-9

Using the microcapsules obtained in Examples 7 to 9, the residualamounts of the physiologically active peptide in the microcapsules weredetermined as in Experimental Example 1, except that the microcapsuledose was about 30 mg/kg. The results are shown in Table 4. Table 5 showsthe linear regression models, correlation coefficients and releaseperiods calculated as X-intercepts, which were determined from the datain Table 4 by the same procedure as used in Table 2.

TABLE 4 Residue of Physiologically active peptide (%) Physiologically 11 2 3 4 active Day Week Weeks Weeks Weeks Experimental A 99.3 74.5 51.433.2 24.1 Example 7 Experimental B 87.4 75.0 52.3 32.8 25.1 Example 8Experimental C 89.4 73.6 54.9 37.7 23.4 Example 9

TABLE 5 Physiologically Linear Release active regression Correlationperiod Peptide model Coefficient (weeks) Experimental A Residue (%) =(R² = 4.9 Example 7 97.8−(20.1 × 0.975) no. of weeks) Experimental BResidue (%) = (R² = 5.0 Example 8 93.5−(18.6 × 0.971) no. of weeks)Experimental C Residue (%) = (R² = 4.9 Example 9 94.4−(18.5 × 0.987) no.of weeks)

It is apparent from Tables 4 and 5 that the microcapsules according tothe present invention are characterized by substantially constantrelease of physiologically active peptide and are further characterizedby being substantially free from an initial burst.

Experimental Example 10

Useing the microcapsules obtained in Example 10, the residual amounts ofthe physiologically active peptide in the microcapsules were determinedas in Experimental Example 7. The results are shown in Table 6. Table 7shows the linear regression models, correlation coefficients and releaseperiods calculated as X-intercepts, which were determined from the datain Table 6 by the same procedure as used in Table 2.

TABLE 6 Residue of physiologically active peptide D (%) Day Week WeekWeek Week 1 1 2 3 4 Experimental 93.5 ± 0.5 69.9 ± 3.6 37.3 ± 1.6 17.0 ±1.4 7.9 ± 0.5 Example 10

TABLE 7 Release Correlation periods Linear regression model coefficient(weeks) Experimental Residue (%) = (R² = 3.9 Example 10 95.0−(24.1 × no.of 0.969) weeks)

It is apparent from Tables 6 and 7 that the microcapsules according tothe present invention are characterized by substantially constantrelease of physiologically active peptide and are further characterizedby being substantially free from an initial burst.

Experimental Example 11

About 30 mg of the microcapsules obtained in Example 11 were dispersedin 0.5 ml of a dispersion medium (prepared by dissolvingcarboxymethylcellulose (2.5 mg), polysorbate 80 (0.5 mg) and mannitol(25 mg) in distilled water) and, the dispersion was injectedsubcutaneously at the back of 10-week-old male SD rats using a 422Gneedle (the dosage as microcapsules 60 mg/kg). Serially afteradministration, the rats were sacrificed, the remains of microcapsuleswere taken out from the administration site and the amount of thephysiologically active peptide A in the microcapsules was determined.The results are shown in Table 8.

Experimental Example 12

Using the microcapsules obtained in Example 12, the procedure ofExperimental Example 11 was otherwise repeated and the residue ofphysiologically active peptide A was assayed. The results are shown inTable 8.

Experimental Example 13

Using the microcapsules obtained in Example 13, the procedure ofExperimental Example 11 was otherwise repeated and the residue ofphysiologically active peptide A was assayed. The results are shown inTable 8.

Experimental Example 14

Using the microcapsules obtained in Example 14, the procedure ofExperimental Example 11 was otherwise repeated and the residue ofphysiologically active peptide A was assayed. The results are shown inTable 8.

TABLE 8 Residue of physiologically active peptide A (%) Day Week WeekWeek Week Week Week 1 1 2 3 4 6 8 Experimental 82.8 21.8 — — — — —Example 11 Experimental 96.7 91.7 79.5 69.2 59.2 — 22.8 Example 12Experimental 100.0 84.3 43.9 31.9 — — — Example 13 Experimental 96.367.5 38.0 23.5 — — — Example 14 (—: not determined)

Table 9 shows the linear regression models, correlation coefficients,and release periods as X-intercept which were determined from the datain Table 8 by the same procedures as used in Table 2.

TABLE 9 Release Correlation periods Linear regression model coefficient(weeks) Experimental Residue (%) = (R² = 1.3 Example 11  97.1−(75.7 ×no. of 0.994) weeks) Experimental Residue (%) = (R² = 10.3  Example 12 92.2−(9.7 × no. of 0.998) weeks) Experimental Residue (%) = (R² = 4.1Example 13 102.4−(24.8 × no. of 0.982) weeks) Experimental Residue (%) =R² = 3.7 Example 14  97.7−(26.5 × no. of 0.989) weeks)

It is apparent from Tables 8 and 9 that the sustained-releasepreparation according to the present invention invariably insure asubstantially constant release of the peptide over various segments ofthe time.

Comparative Example 1

400 mg of physiologically active peptide A acetate was added to asolution of a lactic acid-glycolic acid copolymer ((lactic acid/glycolicacid=50/50 (mole %), GPC weight average mol. wt.=58,000, GPC numberaverage mol. wt.=14,000, number average mol. wt. by end-groupdetermination=45,000; manufacturer; Boehringer-Ingelheim (Lot.RG505-05077), 3.6 g, in 33.2 g (25.0-ml) of dichloromethane but thephysiologically active peptide A acetate could not be successfullydissolved.

Comparative Example 2

400 mg of physiologically active peptide A acetate was added to asolution of lactic acid-glycolic acid copolymer (lactic acid/glycolicacid=75/25 (mole %), GPC weight average mol. wt.=18,000, GPC numberaverage mol. wt.=8,400, number average mol. wt. by end-groupdetermination=30,000; manufacturer; Boehringer-Ingelheim (Lot.RG752-15057), 3.6 g, in 8.0 g (6.0 ml) of dichloromethane but thephysiologically active peptide A could not be successfully dissolved.This dispersion was cooled to 17° C. and poured into 1,000 ml of a 0.1%aqueous solution of polyvinyl alcohol previously adjusted to 15° C. toprepare microcapsules in the same manner as in Example 11. The particlesize distribution and physiologically active peptide A content of themicrocapsules were 10 to 90 μm and 2.5% (w/w), respectively.

Comparative Example 3

400 mg of physiologically active peptide A acetate, was added to asolution of lactic acid-glycolic acid copolymer (lactic acid/glycolicacid=75/25 (mole %), GPC weight average mol. wt.=58,000, GPC numberaverage mol. wt.=15,000, number average mol. wt. by end-groupdetermination=53,000; manufacturer; Boehringer-Ingelheim (Lot.RG755-05019), 3.6 g, in 21.2 g (16.0 ml) of dichloromethane but thephysiologically active peptide A could not be successfully dissolved.This dispersion was cooled to 17° C. and poured into 1,000 ml of a 0.1%aqueous solution of polyvinyl alcohol previously adjusted to 16° C. toprepare microcapsules in the same manner as in Example 11. The particlesize distribution and physiologically active peptide A content of themicrocapsules were 10 to 90 μm and 3.6% (w/w), respectively.

As shown in Comparative Examples 1 to 3, with a lactic acid-glycolicacid copolymer having substantially no terminal carboxyl group, thepeptide [I] of the present invention could not be successfullydissolved.

Comparative Example 4

Leuprorelin acetate (manufacturer: Takeda Chemical Industries), 400 mg,was added to a solution of the same lactic acid-glycolic acid copolymeras used in Comparative Example 2, 3.6 g, in 8.0 g (6.0 ml) ofdichloromethane but the leuprorelin acetate could not be successfullydissolved.

The sustained-release preparation of the present invention shows aconstant release of the drug, especially the peptide [I] over a longtime, thus being conducive to a lasting and stable effect. Furthermore,the duration of release of the drug can be easily controlled andexcessive release immediately following administration can be inhibited.Specifically the histamine-releasing activity in the peptide [I]following administration of the sustained-release preparation isinhibited. The sustained-release preparation has excellentdispersibility. Moreover, the preparation is stable (e.g. to light,heat, humidity, colouring) and of low toxicity and, therefore, can besafely administered.

In accordance with the production method of the present invention, asustained-release preparation containing a physiologically activepeptide can be easily obtained in good yield. The thus obtainedsustained-release preparation has a smooth surface and is excellent inmobility.

What is claimed is:
 1. A method of producing a plurality ofmicrocapsules together constituting a sustained-release preparation ofleuprorelin which comprises: (a) dissolving or suspending leuprorelin inan organic solvent solution comprising an organic solvent selected fromthe group consisting of halogenated hydrocarbons, alkyl ethers havingthree or more carbon atoms, alkyl esters of carboxylic acids wherein thealkyl group has four or more carbon atoms, aromatic hydrocarbons andmixtures thereof and a biodegradable polymer comprising a copolymer oflactic acid and glycolic acid to form a mixture; (b) adding the mixtureto an aqueous medium to provide an O/W emulsion; and (c) transformingthe mixture into microcapsules by removal of the organic solvent.
 2. Themethod of claim 1 wherein said leuprorelin is in the form of leuprorelinacetate.
 3. The method of claim 2 wherein the organic solvent of step(a) comprises dichloromethane.
 4. The method of claim 3 wherein saidaqueous medium of step (b) comprises polyvinyl alcohol in water.
 5. Amethod of producing a plurality of microcapsules together constituting asustained-release preparation of leuprorelin which comprises (a)dissolving or suspending leuprorelin acetate in a dichloromethanesolution of a biodegradable polymer comprising a copolymer of lacticacid and glycolic acid to form a mixture; (b) adding the mixture to anaqueous medium comprising polyvinyl alcohol in water to provide an O/Wemulsion; and (c) transforming the mixture into microcapsules by removalof the dichloromethane solvent.
 6. A method of producing asustained-release preparation which comprises: preparing an oil phasecomprising (a) leuprorelin, (b) a biodegradable lactic acid-glycolicacid copolymer and (c) at least one organic solvent selected from thegroup consisting of halogenated hydrocarbons, alkyl ethers having threeor more carbon atoms, alkyl esters of carboxylic acids wherein the alkylgroup has four or more carbon atoms, aromatic hydrocarbons and mixturesthereof; providing an aqueous phase; forming an O/W emulsion byemulsifying said oil phase in said aqueous phase; and recovering thesustained-release preparation from said emulsion.
 7. The method of claim6, wherein said organic solvent is dichloromethane.
 8. The method ofclaim 6, wherein said leuprorelin is in the form of leuprorelin acetate.9. The method of claim 6 or claim 7, wherein said oil phase is ahomogeneous oil phase.
 10. The method of claim 6 or claim 7, wherein thestep of recovering includes a step of removing at least one organicsolvent from the oil phase.
 11. The method of claim 6 or claim 7,wherein said aqueous phase comprises polyvinyl alcohol and water. 12.The method of claim 6 or claim 7, wherein the lactic acid-glycolic acidcopolymer has a number-average molecular weight determined by end groupdetermination which is about 0.4 to about 2.0 times a number-averagemolecular weight determined by gel permeation chromatography.
 13. Amethod of forming a sustained-release preparation comprising the stepsof: forming an oil phase by adding leuprorelin to a solution comprisinglactic acid-glycolic acid copolymer and at least one organic solventselected from the group consisting of halogenated hydrocarbons, alkylethers having three or more carbon atoms, alkyl esters of carboxylicacids wherein the alkyl group has four or more carbon atoms, aromatichydrocarbons and mixtures thereof; providing an aqueous phase; formingan O/W emulsion by emulsifying said oil phase in said aqueous phase; andrecovering the sustained-release preparation from the emulsion.
 14. Themethod of claim 13, wherein said organic solvent is dichloromethane. 15.The method of claim 13, wherein said leuprorelin is in the form ofleuprorelin acetate.
 16. The method of claim 13 or claim 14, wherein theaqueous phase comprises polyvinyl alcohol and water.
 17. The method ofclaim 13 or claim 14, wherein the lactic acid-glycolic acid copolymerhas a number-average molecular weight determined by end groupdetermination which is about 0.4 to about 2.0 times a number-averagemolecular weight determined by gel permeation chromatograhy.
 18. Themethod of claim 13 or claim 14, wherein the step of recovering includesa step of removing organic solvent from the oil phase.
 19. The method ofclaim 13 or 14, wherein said oil phase is a homogeneous oil phase.